Pcrtanika 5(1),123-125 (1982)

COMMUNICATION (IV)

Enzymes of Hevea brasiliensis latex. Adenylate Kinase, SulphateAdenylyitransferase (ATP-sulphurylase) and Thiosulphate

Sulphurtransferase (Rhodanese)

RINGKASAN

Penyiasatan fasa serum dari lateks Hevea brasiliensis mendalilkan kewujudan ketiga enzim yangberiku t: adenilat kinase EC 2.7.4.3; sulfat adenililtransferase (ATP-sulfurilase) EC 2.7.7.4; tiosulfat sulfur-transferase (rodanese) EC 2.8.1.1.

INTRODUCTION

The latex within the vessels of Hevea brasi-liensis is cytoplasmic and the material obtained bytapping the tree is an exceptionally attractiveobject for biochemical study. It contains numerousorganelles of several kinds, the main ones beingru bber particles, lu toids and Frey-Wyssling com-plexes. These are all suspended in a fluid knownconven tionally as latex serum and correspondingwith the cytosol in the latex vessels (see review byGomez and Moir, 1979). Many enzymes have beenstudied in Hevea latex, for ex ample those involvedin glycolysis (d'Auzac and Jacob, 1969; Bealing,1969) and ru bber biosyn thesis (Archer and Au dley,1967; Lynen, 1969) and the acid hydrolasesoccurring in the lutoids, which identify theseorganelles as a kind of lysosome (Pujarniscle,1968, 1971). Recen t advances inclu de the findingthat two basic proteins characteristic of lutoidsare lysozymes (Tata et al., 1976) and the purifica-tion of a magnesium-dependent phosphatase fromthe serum phase (Souciet et al., 1980).

An unpu blished survey by Dr B. G. Au dley andone of us (G.F.J.M.) shows that seventy enzymeshave been described in Hevea brasiliensis latex.This communication reports the detection of thethree further enzymes listed in the title.

MATERIALS AND METHODS

Latex was collected from Hevea brasiliensistrees of clone RRIM 600 growing in 'Ladang 8'of the farm of Universiti Pertanian Malaysia. Thesetrees had been planted in about 1967; at the timeof the work described here they were being tappedon the second half-spiral panel (Panel B), threetimes a week (Tuesday, Thursday, Saturday).After each tree was tapped the latex was run towaste for 3 min, then collected for 30 min into a

123

flask in an ice bath. The latex from nine trees waspooled and a portion centrifuged at 2° for 40 minat 25,000 r.p.m. (58,000 gmax) in rotor No 65 ofa Beckman L5-65 ultracentrifuge. The latexseparated into the rubber cream, a sediment('bottom fraction') and the serum phase ('C serum')essentially as described by Moir (1959). The tubeswere punctured to recover the C serum: the lower,less turbid portion was used.

Adenylate kinase, EC 2.7.4.3, was assayed bythe method of Aminuddin (1974). The reactionmixture (1 ml) contained TRIS-HCl (35 pmol;pH 7.5), MgCl2 (0.5 pmol), ADP (0.4 pmol) andC serum (0.05 ml). It was incubated for one minat 30° after starting the reaction by adding ADP.1. 0 ml 5% (v Iv) perchloric acid was then added;after centrifugation for 5 min at about 3000 g0.1 ml of the supernatant was mixed with 1.9 mlof ice-cold water and ATP determined in an aliquotof the diluted sample using the continuous bio-luminescence assay method of Balharry andNicholas (1971) as modified by Aminuddin andKooi (1980).

Sulphate adenylyltransferase (ATP-sulphury-lase), EC 2.7.7.4, was assayed by the continuousbioluminescence method of Balharry and Nicholas(1971) as modified by Aminuddin and Kooi(1980).

ThiQSulphate sulphurtransferase (rhodanese),EC 2.8.1.1, was assayed by measuring the thio-cyanate formed from cyanide and thiosulphateaccording to the method of Sorbo (1955) withsome modifications. The substrates were bufferedin 0.1 M borate, pH 10.5; substrates and C serumwere equilibrated at 47° before being mixed amIincu bated at the same temperature for 10 min.The reaction was stopped by the addition of35% (w/w) formaldehyde, followed by the ferric

K. AMPON, M. AMINUDDlN, S. Y. OH, AND G.F.J. MOIR

nitrate reagent for colour development; in thecontrol, formaldehyde was added to the su bstratesbefore C serum.

Protein was estimated in C serum directly,without prior precipitation, using the method ofLowry et al. (1951) with bovine serum albuminas standard. Brzozowska et al. (1974) havereported the presence of some free aromatic aminoacids in C serum, while Tata (1980) has shownthat the use of bovine serum albumin as a standardwhen the Lowry method is applied to C serumproteins results in values considera bly above thetrue level. On both counts, therefore, the proce-dure we used overestimates the protein content ofC serum. Nevertheless it was considered adequatefor the purposes of this preliminary study.

RESULTS AND DISCUSSION

All three enzymes were found in the C serumfrom latex. Table 1 shows typical figures for theiractivities.

We have not yet investigated any differencesin levels of activity that may occur between clonesor with season. We are mainly concerned here withthe occurrence of these enzymes in latex. However,the ATP-sulphurylase has been studied further: theresults will be published separately.

The thiosulphate sulphurtransferase is notablystable. At 57° the enzyme in C serum had almosttwice the activity shown in the assays at 47°.There also seemed to be no loss of activity in Cserum kept frozen for a week. In these propertiesthe enzyme resem bles the thiosulphate sulphur-transferase reported by Chew and Boey (1972) inthe leaves of tapioca (Manihot utilissima). H.brasiliensis and M. utilissima both belong toEuphorbiaceae and both contain the same cyano-genic glycoside, linamarin (Gorter, 1912).

ACKNOWLEDGEMENTS

We thank Dr Idris Abdol, Director of theUniversity Farm, and his staff for supplying theru bber trees an d for help in tapping them. We alsothank Cik Chew Mee Li, Encik Arifen Kamarud-zaman and Encik Mior Ahmad Alang Mior fortheir skilled technical assistance and Cik Hafizahbte Abdul Kadir for typing the manuscript.

K. AmponM. AminuddinSaw-Yin OhG.F.J. Moir

Department of Biochemistry and Microbiology,Faculty of Science and Environmental Studies,Universiti Pertanian Malaysia,Serdang.

TABLE 1

Enzyme activities and protein con tent of C serum of latex.

All measurements on each sample were made on the day the latex was collected. Dates of collection arc shown. Valuesare means of duplicate or triplicate determinations except for ATP-sulphurylase where four and five determinations wereaveraged for samples 1 and 2 respectively.

Enzymes detected

Sulphate adenylyltransferase(ATP-su1phurylase)

Adenyla te kinase

Thiosulphatc sulphurtransferase(Rhodanese)"

1(28/10/80)

39

196

16.8

124

C serum samples2

(4{11{80)

Activity in pkat/mg/protein

41

187

55

Protein content in mg/ml C serum

15.5

3(l1{11{80)

64

16.3

ENZYMES OF HEVEA BRASILIENSIS LATEX

REFERENCES

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AMINUDDIN, M. and KOOI, E.T. (1980): Adenosine-5'triphosphate sulphurylasc from rice shoots: partialpurification und properties. Patanika, 3, 32-39.

ARCHER, B.L. and AUDLEY, E.G. (1967): Biosynthesisof rubber. Adv. Enzymol., 29,221-257.

d'AUZAC, J. and JACOB, J-L. (\969): Regulation ofglycolysis in latex of Hevea brasiliensis. J. Rubb.Res. Inst. Malaya, 21,417-444.

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LOWRY, a.H., ROSEBROUGH, N.J., FARR, A.L. andRANDALL, R.J. (1951): Protein measurement withthe Folin phenol reagen!. J. BioI. ehem., 193 265-275. '

LYNEN, [c. (1969): Biochemical problems of rubbersynthesis. J. Rubb. Res. Inst. Mdtll,l. 21, 389-406.

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PUJARNISCLE, S. (1968): Caractere lysosomal deslutoides ~u latex d'Hevea brasiliensis Mull-Arg.PhySIO. Veg., 6,27-46.

PUJARNISCLE, S. (1971): Etude biochimique deslutoides du latex d'Hevea brasiliensis, Mull. Arg.Differences et analogies avec les lysosomes. MemoireO.R.S.T.O.M. No. 48. Paris: O.R.S.T.O.M. (Office dela Recherche Scientifique et Technique Outre-Mer).

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SOUCIET, G., ATTIAS, J. and d'AUZAC, J. (1980): Aneutral cytoplasmic phosphatase from the latex ofHevea brasiliensis. Phytochem., 19,2099-2102.

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(Received 25 March I 98 I)